Multistrip Western blotting to increase quantitative data output
Multistrip Western blotting to increase quantitative data output
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DOI：
10.1002/elps.200700002
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发表时间：
2007-09-01
期刊：
影响因子：
2.9
通讯作者：
Kiyatkin, Anatoly
中科院分区：
文献类型：
--
作者：
Aksamitiene, Edita;Hoek, Jan B.;Kiyatkin, Anatoly
蛋白质丰度和蛋白质修饰状态的定性和定量测量对于理解它们在不同细胞过程中的作用至关重要。传统的蛋白质印迹技术虽然敏感，但容易产生大量错误，并且不容易适应高通量技术。我们提出了一种改进的免疫印迹程序，这是基于同时转移的蛋白质从多个gelstrips到同一个膜上，并与任何传统的凝胶电泳系统兼容。因此，每个印迹循环的数据输出可以容易地增加高达十倍。与传统的“每个电泳周期检测一种蛋白质”不同，该程序允许同时监测多达9种不同的蛋白质。除了保持检测皮克数量的蛋白质的能力之外，修改后的系统通过将信号误差减少两倍来显著提高数据准确性。多条带蛋白质印迹法允许对不同或重复的数据集进行统计学上可靠的并排比较。与传统方法相比，该方法提供了一种更经济、可重复和有效的方法，便于产生大量高质量的可量化数据。
The qualitative and quantitative measurements of protein abundance and protein modification states are essential in understanding their role in diverse cellular processes. Traditional Western blotting technique, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. We propose a modified immunoblotfing procedure, which is based on simultaneous transfer of proteins from multiple gelstrips onto the same membrane, and is compatible with any conventional gel electrophoresis system. As a result, the data output per single blotting cycle can readily be increased up to ten-fold. In contrast to the traditional "one protein detection per electrophoresis cycle", this procedure allows simultaneous monitoring of up to nine different proteins. In addition to maintaining the ability to detect picogram quantities of protein, the modified system substantially improves data accuracy by reducing signal errors by two-fold. Multistrip Western blotting procedure allows making statistically reliable side-by-side comparisons of different or repeated sets of data. Compared to the traditional methods, this approach provides a more economical, reproducible, and effective procedure, facilitating the generation of large amounts of high-quality quantifiable data.